微孔板与标准平皿Ames试验比较研究
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摘要
目的使用鼠伤寒沙门菌和大肠埃希菌及阳性剂开展基于6孔板、24孔板和标准10 cm平皿的Ames试验,为确立标准化微孔板Ames试验奠定研究基础。方法 6种不同鼠伤寒沙门菌(TA97、TA98、TA100、TA102、TA1535、TA1537)和1种大肠埃希菌(WP2 uvr A)经鉴定及扩增后,分别使用标准平皿、6孔板和24孔板在有无S9代谢活化条件下使用不同浓度的阳性剂开展平板掺入法Ames试验。所有样本置37℃培养约48 h后进行菌落计数。结果所有试验条件下均出现浓度相关性的回复菌落形成率增加,且最高浓度条件下的菌落数高于对照组3倍。然而微孔板中可出现阴性背景菌落数过少的情况,阳性剂的用量也不能照搬标准平皿中的浓度设置。结论本研究所用条件可成功开展基于6孔板和24孔板的Ames试验,其试验条件和背景数据需要经过摸索与积累。
Objectives To compare the Ames tests based on 6-well/24-well plates and standard 10 cm plates using Salmonella typhimurium/Escherichia coli and postive agents, and lay a research foundation for the standardized microplate based Ames tests.Methods 6 Salmonella typhimurium(TA97, TA98, TA100, TA102, TA1535, TA1537) and a Escherichia coli(WP2 uvrA) were idenfied and amplified, and subsequently used to perform standard plate, 6-well plate and 24-well plate based Ames tests with a range of different concentrations of positive agents in the presence or absence of S9 metabolic activation. The colonies were counted48 hours after cultured at 37 ℃. Results The concentration relevant colony formation rates increasing were observed in all test conditions Under the highest concentrations, the colony counts were 3 times higher than the solvent control. However, too few backgournd colony counts might be found in the solvent controls of microplates, and the amount of positive agents might not be set in accord with the concentrations used in standard plate test. Conclusions 6-well and 24-well plate based Ames tests were performed successfully following the current protocol, whereas the experimental conditions and background data shall be further explored and accumulated.
Objectives To compare the Ames tests based on 6-well/24-well plates and standard 10 cm plates using Salmonella typhimurium/Escherichia coli and postive agents, and lay a research foundation for the standardized microplate based Ames tests.Methods 6 Salmonella typhimurium(TA97, TA98, TA100, TA102, TA1535, TA1537) and a Escherichia coli(WP2 uvrA) were idenfied and amplified, and subsequently used to perform standard plate, 6-well plate and 24-well plate based Ames tests with a range of different concentrations of positive agents in the presence or absence of S9 metabolic activation. The colonies were counted48 hours after cultured at 37 ℃. Results The concentration relevant colony formation rates increasing were observed in all test conditions Under the highest concentrations, the colony counts were 3 times higher than the solvent control. However, too few backgournd colony counts might be found in the solvent controls of microplates, and the amount of positive agents might not be set in accord with the concentrations used in standard plate test. Conclusions 6-well and 24-well plate based Ames tests were performed successfully following the current protocol, whereas the experimental conditions and background data shall be further explored and accumulated.
引文
[1] Mortelmans K, Zeiger E. The Ames Salmonella/microsome mutagenicity assay[J]. Mutat Res, 2000, 455(1):29-60.
[2]于仲波,吴南翔,金锋,等. 1485种化学物致突变试验和致癌试验结果一致性比较[J].毒理学杂志, 2007(4):320-320.
[3] Cassano A, Raitano G, Mombelli E, et al. Evaluation of QSAR Models for the prediction of Ames genotoxicity:A retrospective exercise on the chemical substances registered under the EU REACH regulation[J]. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev, 2014, 32(3):273-298.
[4] ISO10993-3:2003 Biological evaluation of medical devices-Part 3:Tests for genotoxicity, carcinogenicity and reproductive toxicity[S]. 2003.
[5] GB 15193.4-2014,食品安全国家标准细菌回复突变试验[S]. 2014.
[6] ICH. Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use S2(R)[EB/OL].(2011-11-9)[2018-7-2]. http://www.ich. org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Safety/S2_R1/Step4/S2R1_Step4.pdf.
[7]药物遗传毒性研究技术指导原则课题研究组.药物遗传毒性研究技术指导原则2018[EB/OL].(2018-3-12)[2018-7-2]. http://www.sdfda.gov.cn/art/2018/3/15/art_6088_200748.html.
[8] Gee P, Maron D M, Ames B N. Detection and classification of mutagens:a set of base-specific Salmonella tester strains[J]. Proc Natl Acad Sci U S A,1994, 91(24):11606-11610.
[9]文海若,宋捷,刘倩,等. Ames波动试验和GADD45aGFP GreenScreen两种快速筛选方法评价大黄素和芫花素的遗传毒性[J].药物评价研究, 2018(5):734-741.
[10] Zwart N, Lamoree M, Houtman C, et al. Development of a luminescent mutagenicity test for high-throughput screening of aquatic samples[J]. Toxicol In Vitro, 2018(46):350-360.
[11]许雷鸣,胡燕平,武谷,等.现行药物、保健食品和化妆品Ames试验方法的比较分析[J].安徽医药, 2013, 17(9):1595-1597.
[12]胡燕平,宋捷,李波.细菌回复突变试验背景数据的采集[J].中国新药杂志, 2009(22):2110-2112.
[13] Sugiyama K, Yamada M, Awogi T, et al. The strains recommended for use in the bacterial reverse mutation test(OECD guideline 471)can be certified as nongenetically modified organisms[J]. Genes Environment,2016, 38(1):1-3.
[14] Gatehouse D. Bacterial mutagenicity assays:test methods[J]. Methods Mol Biol, 2012, 817:21-34.
[15] Flamand N, Meunier J R, Meunier P A, et al. Mini mutagenicity test:a miniaturized version of the Ames test used in a prescreening assay for point mutagenesis assessment[J]. Toxicol In Vitro, 2001, 15(2):105-114.
[16] Wilson J D, Cariello N F. The Ames miniscreen assay:Volatility of sodium azide can cause an increase in the reversion frequencies of adjacent, untreated wells[J].Environ Mol Mutagen, 1997, 29(2):217-219.
[17] Cooper A L, Dean A C, Hinshelwood C. Factors affecting the growth of bacterial colonies on agar plates[J]. Proc R Soc Lond B Biol Sci, 1968, 171(23):175-199.
[18] Zeiger E. Carcinogenicity of mutagens:predictive capability of the Salmonella mutagenesis assay for rodent carcinogenicity[J]. Cancer Res, 1987, 47(5):1287-1296.
[19] Liberman D F, Fink R C, Schaefer F L, et al.Mutagenicity of anthraquinone and hydroxylated anthraquinones in the Ames/Salmonella microsome system[J]. Appl Environ Microbiol, 1982, 43(6):1354.
[20] Grey C E, Adlercreutz P. Ability of antioxidants to prevent oxidative mutations in Salmonella typhimurium TA102[J]. Mutat Res, 2003, 527(1):27-36.
[21] Prival M J, Zeiger E. Chemicals mutagenic in Salmonella typhimurium strain TA1535 but not in TA100[J]. Mutat Res, 1998, 412(3):251-260.
[22] ICH. OECD Guidelines for the Testing of Chemicals TG471:Bacterial Reverse Mutation Test[EB/OL].(1997-7-21)[2018-7-2]. https://www. oecd-ilibrary. org/environment/test-no-471-bacterial-reverse-mutation-test_9789264071247-en.
[2]于仲波,吴南翔,金锋,等. 1485种化学物致突变试验和致癌试验结果一致性比较[J].毒理学杂志, 2007(4):320-320.
[3] Cassano A, Raitano G, Mombelli E, et al. Evaluation of QSAR Models for the prediction of Ames genotoxicity:A retrospective exercise on the chemical substances registered under the EU REACH regulation[J]. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev, 2014, 32(3):273-298.
[4] ISO10993-3:2003 Biological evaluation of medical devices-Part 3:Tests for genotoxicity, carcinogenicity and reproductive toxicity[S]. 2003.
[5] GB 15193.4-2014,食品安全国家标准细菌回复突变试验[S]. 2014.
[6] ICH. Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use S2(R)[EB/OL].(2011-11-9)[2018-7-2]. http://www.ich. org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Safety/S2_R1/Step4/S2R1_Step4.pdf.
[7]药物遗传毒性研究技术指导原则课题研究组.药物遗传毒性研究技术指导原则2018[EB/OL].(2018-3-12)[2018-7-2]. http://www.sdfda.gov.cn/art/2018/3/15/art_6088_200748.html.
[8] Gee P, Maron D M, Ames B N. Detection and classification of mutagens:a set of base-specific Salmonella tester strains[J]. Proc Natl Acad Sci U S A,1994, 91(24):11606-11610.
[9]文海若,宋捷,刘倩,等. Ames波动试验和GADD45aGFP GreenScreen两种快速筛选方法评价大黄素和芫花素的遗传毒性[J].药物评价研究, 2018(5):734-741.
[10] Zwart N, Lamoree M, Houtman C, et al. Development of a luminescent mutagenicity test for high-throughput screening of aquatic samples[J]. Toxicol In Vitro, 2018(46):350-360.
[11]许雷鸣,胡燕平,武谷,等.现行药物、保健食品和化妆品Ames试验方法的比较分析[J].安徽医药, 2013, 17(9):1595-1597.
[12]胡燕平,宋捷,李波.细菌回复突变试验背景数据的采集[J].中国新药杂志, 2009(22):2110-2112.
[13] Sugiyama K, Yamada M, Awogi T, et al. The strains recommended for use in the bacterial reverse mutation test(OECD guideline 471)can be certified as nongenetically modified organisms[J]. Genes Environment,2016, 38(1):1-3.
[14] Gatehouse D. Bacterial mutagenicity assays:test methods[J]. Methods Mol Biol, 2012, 817:21-34.
[15] Flamand N, Meunier J R, Meunier P A, et al. Mini mutagenicity test:a miniaturized version of the Ames test used in a prescreening assay for point mutagenesis assessment[J]. Toxicol In Vitro, 2001, 15(2):105-114.
[16] Wilson J D, Cariello N F. The Ames miniscreen assay:Volatility of sodium azide can cause an increase in the reversion frequencies of adjacent, untreated wells[J].Environ Mol Mutagen, 1997, 29(2):217-219.
[17] Cooper A L, Dean A C, Hinshelwood C. Factors affecting the growth of bacterial colonies on agar plates[J]. Proc R Soc Lond B Biol Sci, 1968, 171(23):175-199.
[18] Zeiger E. Carcinogenicity of mutagens:predictive capability of the Salmonella mutagenesis assay for rodent carcinogenicity[J]. Cancer Res, 1987, 47(5):1287-1296.
[19] Liberman D F, Fink R C, Schaefer F L, et al.Mutagenicity of anthraquinone and hydroxylated anthraquinones in the Ames/Salmonella microsome system[J]. Appl Environ Microbiol, 1982, 43(6):1354.
[20] Grey C E, Adlercreutz P. Ability of antioxidants to prevent oxidative mutations in Salmonella typhimurium TA102[J]. Mutat Res, 2003, 527(1):27-36.
[21] Prival M J, Zeiger E. Chemicals mutagenic in Salmonella typhimurium strain TA1535 but not in TA100[J]. Mutat Res, 1998, 412(3):251-260.
[22] ICH. OECD Guidelines for the Testing of Chemicals TG471:Bacterial Reverse Mutation Test[EB/OL].(1997-7-21)[2018-7-2]. https://www. oecd-ilibrary. org/environment/test-no-471-bacterial-reverse-mutation-test_9789264071247-en.